Epinephrine compositions and containers

ABSTRACT

The inventive subject matter provides ready-to-administer, preferably anti-oxidant free, epinephrine compositions with improved stability, and methods for preparing the same. Contemplated compositions can be packaged using blow-fill-seal technology or packaged into flexible IV bags and maintain degradation of the epinephrine at a level of less than 5 wt % when stored over at least one months at between 2-40° C.

This application is a continuation application of our copending U.S.application with the Ser. No. 16/788,831, filed Feb. 12, 2020, which isa continuation application of our allowed U.S. application with the Ser.No. 16/360,995, filed Mar. 21, 2019, and which claims priority to USprovisional application with the Ser. No. 62/647,303, filed Mar. 23,2018.

FIELD OF THE INVENTION

The field of the invention is pharmaceutical compositions and methods ofmanufacture of same, especially as they relate to storage stable andready-to-inject epinephrine compositions.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Epinephrine is an endogenous adrenergic neurotransmitter secreted by themedulla of the adrenal glands and acts on both alpha and beta adrenergicreceptors found ubiquitously throughout much of the human body. As suchepinephrine is involved in various regulatory processes, includingregulation of heart beat, blood pressure, airway resistance, and energymetabolism.

Epinephrine, USP is a sympathomimetic catecholamine (adrenergic agent)designated chemically as4-[1-hydroxy-2(methylamino)ethyl]-1,2-benzenediol, a white,microcrystalline powder and has the following structural formula:

Currently, epinephrine is marketed as a concentrated form for injection(1 mg/mL), which must be diluted with a proper diluent such as dextroseor dextrose/NaCl. Unfortunately, all or almost all of the known dilutedepinephrine formulations that are commercially available lack storagestability. Indeed generally, diluted epinephrine solutions must bediscarded within one day after reconstitution when stored at roomtemperature. Since diluted epinephrine solutions are unstable withlimited shelf life, such solutions are almost never sold or shipped, butmust be prepared at hospitals or doctor's offices for immediate use.Consequently, currently known concentrated epinephrine forms carry asubstantial risk of dilution errors, while diluted forms carry a risk ofmicrobial contamination and often have very limited shelf-life due toreduced chemical stability.

Epinephrine in aqueous solution deteriorates rapidly on exposure to airor light (typically via auto-oxidation), turning pink from oxidation toadrenochrome and turning brown from formation of melanin. The rate ofthis reaction increases with increased pH, increased temperature, and inthe presence of metal ions such as aluminum from various rubbers andiron from amber glass ware. Epinephrine solutions may also lose potencyas a result of racemization from the biologically active R-isomer to thebiologically inactive S-isomer. Such racemization notably increases withincreasing temperatures. Epinephrine is also rapidly destroyed inalkaline solutions by aldehydes, weak oxidizing agents, and atmosphericoxygen. Epinephrine solutions can be stored under refrigeration toreduce degradation, but longer-term stability at reduced temperaturenevertheless remains a problem. Additionally, availability ofrefrigerated storage in doctors' offices and hospital is low, and soepinephrine solutions are not often stored in this manner. Whereasconcentrated epinephrine solutions in small vials take up little room inrefrigerated storage, low concentration solutions most often used forpatients are often in 100 ml or 1000 ml bags, and take up valuable spaceif stored under refrigeration.

Epinephrine can be chemically modified to reduce degradation, however,chemical modification of catecholamines is generally undesirable for anumber of reasons, including significant loss of biological activity. Toovercome difficulties associated with chemical modification,antioxidants such as sodium metabisulfite or sodium bisulfite have beenused to protect catecholamines or adrenergic compounds againstauto-oxidation. For example, where epinephrine and sodium metabisulfitewere used at a ratio of about 1:0.005 to about 1:15 by weight,formulations with desirable storage stability were obtained. However,such antioxidants have been associated in at least some cases withsevere allergic reactions. In addition, sodium bisulfite can reactdirectly with epinephrine in aqueous solution upon exposure to roomlight probably due to the conversion of superoxide (O2-) radicals tohighly reactive hydroxyl (.OH) radicals by bisulfite (see e.g., Photodestabilization of Epinephrine by sodium metabisulfite (PDA J Pharm SciTechnol. 2000 Mar-Apr;54(2):136-43.). In addition, the potency ofepinephrine could be further substantially degraded during shelf lifestorage due to radical-mediated reactions. Therefore, in view of thereactivity of epinephrine with widely used antioxidants, use of suchcompounds is not advisable in epinephrine containing formulations.

In yet other attempts to increase storage stability of epinephrineformulations, inclusion complexes of epinephrine with native or modifiedcyclodextrin derivatives have been prepared as is described in US2018/0028671. While such compositions reduced thermal and/or oxidativedegradation, the use of complexing agents rendered manufacture moredifficult. Moreover, the bioavailability of the epinephrine may bereduced at least in some formulations. Furthermore, when epinephrine isdiluted with water, such water often contains dissolved oxygen, as doesthe head space in the container. The oxygen in the water acts to degradethe epinephrine or other oxygen sensitive drug, increasing degradantsand decreasing shelf life.

Therefore, even though various methods are known in the art to stabilizeoxygen-sensitive drugs and specifically epinephrine in solution,numerous difficulties remain, particularly where epinephrine is presentin an aqueous solution at relatively low concentrations. Consequently,there is a need for improved stable, low concentration,ready-to-administer antioxidant free epinephrine formulations, andmethods of manufacture and storage of such formulations.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to antioxidant freesterilizable/autoclavable and ready-to-administer compositionscontaining an oxygen-sensitive drug, such as epinephrine, havingimproved stability and a physiologically acceptable pH. As used herein,reference to the term epinephrine or any other drug should beinterpreted broadly to include all pharmaceutically acceptable salts andprodrug forms thereof. Additionally, the storage systems described herecan be used for solutions of other oxygen-sensitive drugs.

In one aspect of the inventive subject matter, the inventors contemplatean antioxidant-free and storage stable ready-to-administer compositioncontaining an oxygen-sensitive drug, for example, an epinephrinecomposition that includes an aqueous pharmaceutically acceptable carriercontaining epinephrine. Preferably, the epinephrine is present in theready-to-administer epinephrine composition at a concentration of equalor less than about 0.07 mg/ml, and substantially all (i.e., at leastabout 98 mol %) of the epinephrine is an R-isomer. It is furtherpreferred that the ready-to-administer epinephrine composition has a pHof between about 3.0 and about 4.7, and that the ready-to-administerepinephrine composition further comprises a metal ion chelator (e.g.,EDTA, edetic acid, EGTA and diethylenetriaminepentaacetic acid). Infurther preferred aspects, the ready-to-administer epinephrinecomposition has, after storage of at least one month, total impuritiesof equal or less than about 0.7% and equal or less than about 2%S-isomer content.

For example, in some embodiments the epinephrine is present in thecomposition at a concentration of between about 0.005 mg/ml and about0.050 mg/ml, or at a concentration of between about 0.006 and about0.010 mg/ml, or at a concentration of between about 0.010 and about0.025 mg/ml, or at a concentration of between about 0.025 and about0.045 mg/ml. Where desired, the epinephrine is present in thecomposition as a pharmaceutically acceptable salt. In furtherembodiments, the ready-to-administer epinephrine composition has, afterstorage of at least one month at 25° C. +/−2° C., a pH of between about3.5 and about 4.5, or a pH of between about 3.8 and about 4.2.

Most typically, the aqueous pharmaceutically acceptable carrier is waterfor injection, and may further comprise a buffer (e.g., buffer ispresent in a concentration of between about 1 mM and about 25 mM, buffermay be an acetate buffer, a citrate buffer, a phosphate buffer, atartrate buffer, and a borate buffer). Contemplated compositions mayalso include a tonicity agent (e.g., sodium chloride, glycerol,thioglycerol, mannitol, lactose, and dextrose). Additionally, it ispreferred that the ready-to-administer epinephrine composition hasdissolved oxygen of equal or less than 1.5 ppm O₂.

In further embodiments, the ready-to-administer epinephrine compositionhas, after storage of at least one month at 25° C. +/−2° C., totalimpurities of equal or less than about 0.5% and equal or less than about1% S-isomer content, or the ready-to-administer epinephrine compositionhas, after storage of at least one month at 25° C. +/−2° C., totalimpurities of equal or less than about 0.2% and equal or less than about1.5% S-isomer content, or the ready-to-administer epinephrinecomposition has, after storage of at least one month at 25° C. +/−2° C.,total impurities of equal or less than about 0.5% and equal or less thanabout 1% S-isomer content, or the ready-to-administer epinephrinecomposition has, after storage of at least one month at 25° C. +/−2° C.,total impurities of equal or less than about 0.3% and equal or less thanabout 0.7% S-isomer content. Additionally, or alternatively, theready-to-administer epinephrine composition has, after autoclaving,total impurities of equal or less than about 0.5% and equal or less thanabout 2.0% S-isomer content, or has, after autoclaving, total impuritiesof equal or less than about 0.2% and equal or less than about 1.5%S-isomer content.

Therefore, in yet another aspect of the inventive subject matter, theinventors also contemplate a pharmaceutical product, a polymericcontainer that includes the antioxidant-free and storage stableready-to-administer composition containing an oxygen-sensitive drug,such as the epinephrine composition presented herein, wherein thecontainer is a blow-fill-seal (BFS) container or flexible IV bag. Forexample, suitable polymeric containers may have a volume of between 100mL and 1,000 mL, and may be further enclosed in a metallizedover-container. Where desired, an oxygen scavenger or absorber may bedisposed between the polymeric container and the metallizedover-container. In still further contemplated embodiments, metallizedover-container will have an oxygen transmission rate of between 0.0005to 2.00 cc/100 in²/24 hrs.

In still further aspects of the inventive subject matter, the inventorsalso contemplate method of producing a storage stableready-to-administer epinephrine composition that comprises a step ofcombining an aqueous pharmaceutically acceptable carrier withepinephrine in an amount such that the epinephrine is present in theready-to-administer epinephrine composition at a concentration of equalor less than about 0.07 mg/ml. Most typically, the aqueouspharmaceutically acceptable carrier has dissolved oxygen in an amount ofequal or less than about 2 ppm. In another step, the pH of theready-to-administer epinephrine composition is adjusted to a pH ofbetween about 3.0 and about 4.7, and a metal ion chelator (e.g., EDTA,edetic acid, EGTA and diethylenetriaminepentaacetic acid) is includedinto the ready-to-administer epinephrine composition. In a still furtherstep, the ready-to-administer composition containing an oxygen-sensitivedrug, such as epinephrine composition is packaged into a polymericcontainer under an inert gas and the ready-to-administer epinephrinecomposition is sterilized.

For example, the step of sterilizing may comprise autoclaving, typicallyusing saturated steam at 121° C. for at least 10 minutes, and preferablywhen the ready-to-administer composition containing an oxygen-sensitivedrug, such as epinephrine composition is in the polymeric container. Asnoted earlier, the polymeric container may be further packaged into ametallized over-container.

With respect to suitable epinephrine concentrations it is contemplatedthat such concentrations may be between about 0.005 mg/ml and about0.050 mg/ml, or between about 0.006 and about 0.010 mg/ml, or betweenabout 0.010 and about 0.025 mg/ml, or between about 0.025 and about0.045 mg/ml. As will be readily appreciated, the epinephrine may bepresent in the ready-to-administer composition as a pharmaceuticallyacceptable salt.

It is further contemplated that the ready-to-administer epinephrinecomposition has, after storage of at least one month at 25° C. +/−2° C.,a pH of between about 3.5 and about 4.5, or between about 3.8 and about4.2. Preferably, the aqueous pharmaceutically acceptable carrier iswater for injection, which may or may not further include a buffer(e.g., acetate buffer, citrate buffer, phosphate buffer, tartratebuffer, borate buffer, typically at a concentration of between about 1mM and about 25 mM). Where desired, a tonicity agent (e.g., sodiumchloride, glycerol, thioglycerol, mannitol, lactose, dextrose) may beincluded into the ready-to-administer composition. Additionally, oralternatively, the ready-to-administer epinephrine composition hasdissolved oxygen of equal or less than 1.5 ppm O₂.

In some embodiments of contemplated methods, the ready-to-administerepinephrine composition has, after storage of at least one month at 25°C. +/−2° C., total impurities of equal or less than about 0.5% and equalor less than about 1% S-isomer content, or has, after storage of atleast one month at 25° C. +/−2° C., total impurities of equal or lessthan about 0.2% and equal or less than about 1.5% S-isomer content, orhas, after storage of at least one month at 25° C. +/−2° C., totalimpurities of equal or less than about 0.5% and equal or less than about1% S-isomer content. In further embodiments of contemplated methods, theready-to-administer epinephrine composition has, after storage of atleast one month at 25° C. +/−2° C., total impurities of equal or lessthan about 0.3% and equal or less than about 0.7% S-isomer content, orhas, after sterilizing, total impurities of equal or less than about0.5% and equal or less than about 2.0% S-isomer content, wherein thesterilizing is autoclaving, or has, after sterilizing, total impuritiesof equal or less than about 0.2% and equal or less than about 1.5%S-isomer content, wherein the sterilizing is autoclaving.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have discovered that various antioxidant-free and storagestable ready-to-administer compositions containing an oxygen-sensitivedrug, such as epinephrine compositions can be prepared in a relativelysimple and effective manner. Advantageously, the compositions presentedherein have excellent storage stability even over extended periods, andhave suitably low concentrations of epinephrine that allowsadministration of the composition directly to a patient at a very lowdosage rate (e.g., 0.05-2.0 mcg/kg/min) without prior dilution.Moreover, the inventors discovered that the compositions can besterilized (e.g., using autoclaving) without producing undesirablequantities of biologically inactive S-isomers. Thermal stability alsoallows the pharmaceutical compositions to be packaged in ablow-fill-seal (BFS) process. Alternatively, the compositions presentedherein can be filled into flexible infusion bags (e.g., IV).

For example, in one embodiment the antioxidant-free and storage stableready-to-administer epinephrine composition includes an aqueouspharmaceutically acceptable carrier containing epinephrine such thatepinephrine is present in the ready-to-administer epinephrinecomposition at a concentration of equal or less than about 0.07 mg/ml.Moreover, the ready-to-administer epinephrine composition has a pH ofbetween about 3.0 and about 4.7, and further comprises a chelator(typically a metal ion chelator). In further embodiments, theready-to-administer epinephrine composition has, after storage of atleast one month at 25° C. +/−2° C., total impurities of equal or lessthan about 0.7% and equal or less than about 2% S-isomer content.

In still further embodiments, the ready-to-administer epinephrinecomposition is packaged in flexible infusion bags or blow-fill-seal(BFS) containers and is sterilized, preferably by autoclaving. Theinventors further unexpectedly discovered that epinephrine formulationsat a relatively low concentrations and a pH range of about 3.5 and about5.0 can be subjected to terminal sterilization, and particularlysterilizing to sterility (e.g., over at least 5 minutes, or at least 10minutes, or at least 15 min at 121° C.), without substantial increase ofthe S-isomer of epinephrine while being storage stable, especially whenformulated with a metal ion chelator (e.g., EDTA). In furtherembodiments, the BFS container or flexible IV bag is further packaged ina secondary container, optionally with an oxygen scavenger, andespecially a metal free oxygen scavenger. Most typically, at least oneof the polymer bag and the secondary container may be impervious tolight in general or light of a wavelength that promotes photo-initiateddegradation. For example, suitable containers may be metalized (e.g.,aluminized), or combined or coated with various carbonaceous materialsor other dye(s).

In some preferred aspects, the epinephrine composition is aready-to-administer formulation wherein epinephrine is present at aconcentration of between about 0.001 to about 0.07 mg/ml, or betweenabout 0.005 to about 0.07 mg/ml, or between about 0.005 to about 0.05mg/ml, or between about 0.005 to about 0.03 mg/ml, or between about0.005 to about 0.02 mg/ml. Viewed from a different perspective, whilehigher concentration epinephrine compositions are also contemplated,some preferred formulations will include epinephrine at a concentrationof equal or less than about 0.07 mg/ml, equal or less than about 0.05mg/ml, or even equal or less than about 0.02 mg/ml, such that nodilution is required prior to injection. Therefore, epinephrine may bepresent in the ready-to-administer formulation at a concentration ofbetween about 0.005 mg/ml and about 0.050 mg/ml, or at a concentrationof between about 0.006 and about 0.010 mg/ml, or at a concentration ofbetween about 0.010 and about 0.025 mg/ml, or at a concentration ofbetween about 0.025 and about 0.045 mg/ml.

Of course, it should be appreciated that epinephrine may be present inthe composition as base or in form of a pharmaceutically acceptablesalt, including a hydrochloride salt, a bitartrate salt, and a boratesalt as such salt forms substantially increase the solubility ofepinephrine in an aqueous medium. Moreover, it is generally preferredthat substantially all of the epinephrine is in the R-isomer form (e.g.,at least about 85 mol %, or at least about 90 mol %, or at least about95 mol %, or at least about 97 mol %, or at least about 99 mol %). Instill further preferred aspects, the aqueous pharmaceutically acceptablecarrier is water, which may further include one or more polar and/orprotic solvents that will typically form a single-phase solvent systemwith the water.

With further respect to the aqueous pharmaceutically acceptable carrier,it is typically preferred that the carrier and/or theready-to-administer epinephrine composition has a dissolved oxygencontent of equal or less than about 5.0 ppm or equal or less than about3.0 ppm or equal or less than about 2.5 ppm or equal or less than about2.0 ppm or equal or less than about 1.5 ppm or equal or less than about1.0 ppm O₂ (typically during compounding and/or after 1 month of storageat 25° C. +/−2° C.). To that end, the carrier and/or ready-to-administerepinephrine composition can be subjected to sparging with an inert gas(e.g., argon, helium, freons, nitrogen, etc.), vacuum stripping underagitation, storage under an inert gas headspace, storage in a polymericcontainer with metallized over-container that further includes an oxygenabsorber/scavenger (e.g., metal-free oxygen scavenger (e.g., GLS100,Ageless®, Pharmakeep®, all commercially available from Mitsubishi GasChemical America)), and/or using an enzymatic systems that deplete asolution of dissolved oxygen (see e.g., U.S. 9,187,779).

In other aspects of the inventive subject matter, contemplatedready-to-administer epinephrine composition can have a mildly acidic pHof between about 3.0 and about 5.5 or between about 4.0 and about 6.0,such as a pH of between about 3.5 and about 4.5 or a pH of between about3.8 and about 4.2. Most typically the pH of the ready-to-administerepinephrine composition will be less than about 5.0 and more typicallyless than about 4.5, and most typically less than about 4.3, but higherthan about 3.0, more typically higher than about 3.5, and most typicallyhigher than about 3.7. As will be readily appreciated, the pH can beadjusted using various acids (e.g., HCl ) and bases (e.g., NaOH).Moreover, and where desired, the pH can be stabilized using a buffer,typically at a relatively low strength. For example, suitable buffersmay be present at a concentration of between about 1 mM and about 50 mM,or between about 10 mM and about 25 mM, or between about 20 mM and about40 mM. Among other choices, contemplated buffers include organic andinorganic buffers, as well as amphoteric buffers. For example, suitablebuffers include an acetate buffer, a citrate buffer, a phosphate buffer,a tartrate buffer, and a borate buffer, which may be adjusted to a pH inthe range of between about 3.7 and about 4.3, or between about 3.7 andabout 4.0, or between about 3.8 and about 4.1, or between about 3.9 andabout 4.2, or between about 4.0 and about 4.2. Notably, such pH rangesprovided remarkable stability for low concentrations of epinephrine,especially where epinephrine was combined with a chelator and optionaltonicity agents.

Additionally or alternatively, the epinephrine formulations can bepackaged in blow-fill-seal (BFS) containers/flexible IV bags, which arefurther packaged in an aluminum foil pouch or single- or multi-layeroverwrap with an oxygen scavenger (secondary packaging), or aluminumpouches containing an oxygen absorbing layer (secondary packaging),where the outer pouch or packaging has an oxygen transmission rate (OTR)between 0.0005 to 0.005, or between 0.005 to 0.05, or between 0.05 to0.5, or between 0.5 to 2.00, or between 1.0 to 5.00 cc/100 in²/24 hrs.

Moreover, in further contemplated aspects, the ready-to-administerepinephrine composition will also include one or more chelating agents,and particularly metal ion chelators to slow down the baseline and metalion-stimulated autoxidation of epinephrine. For example, suitablechelators include various bicarboxylic acids, tricarboxylic acids, andaminopolycarboxylic acids such as ethylenediaminetetraacetic acid(EDTA), ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraaceticacid (EGTA), and penta(carboxymethyl)diethylenetriamine (DTPA), andsalts and hydrates thereof. While not limiting to the inventive subjectmatter, it is contemplated that the metal ion chelators will slow downboth the baseline and metal ion-stimulated autoxidation of epinephrine.Notably, the inventors unexpectedly observed that the desirable effectof the chelators was observable at relatively low concentrations of thechelators. For example, reduction of the baseline and metalion-stimulated autoxidation of epinephrine was observed at chelatorconcentrations of between about 1 μg/ml and about 10 μg/ml, and betweenabout 10 μg/ml and about 100 μg/ml. Thus, preferred chelatorconcentrations will be between about 1 μg/ml and about 50 μg/ml, orbetween about 5 μg/ml and about 25 μg/ml. Interestingly, the chelators,and especially the aminopolycarboxylic acids retained stabilizing effectdespite the relatively low pH favoring protonated forms of thechelators.

With respect to suitable tonicity agents, pharmaceutically acceptablesalts are generally preferred to adjust/increase tonicity. For example,NaCl may be employed at a concentration of at least about 0.6 wt %, orat least about 0.7 wt %, or at least about 0.8 wt %, or at least about0.9 wt %. Thus, suitable salt concentrations will typically be betweenabout 0.6 wt % and about 1.2 wt %. Depending on the particular saltconcentration, additional tonicity agents may be added and such suitableagents include glycerol, thioglycerol, mannitol, lactose, and dextrose.The amount of tonicity adjusting agent used can be adjusted to obtainosmolality of the formulations, typically in the range of about 260 toabout 340 mOsm/kg. An osmometer can be used to check and adjust theamount of tonicity adjusting agent to be added to obtain the desiredosmolality.

It should further be appreciated that contemplated compositions aresubstantially free of antioxidants (i.e., do not include antioxidants inan amount effective to reduce degradation of total epinephrine by atleast about 1% when stored over a period of at least three months at 25°C. +/−2° C.). Therefore, and viewed from a different perspective,antioxidant-free and storage stable ready-to-administer epinephrinecomposition will include antioxidants in an amount of equal or less thanabout 0.01 wt %, or equal or less than about 0.005 wt %, or equal orless than about 0.001 wt %, or equal or less than about 0.0005 wt %, orequal or less than about 0.0001 wt %. Remarkably, despite the lack ofantioxidants added to the formulation, the ready-to-administerepinephrine composition had unexpected storage stability over extendedperiods with regard to both oxidation/degradation and isomerization. Forexample, in some embodiments, the ready-to-administer epinephrinecomposition has, after storage of at least one month at 25° C. +/−2° C.,total impurities of equal or less than about 0.7% and equal or less thanabout 2% S-isomer content. Moreover, and as is shown in more detailbelow, such storage stability extended to at least 3 months, at least 6months, at least 9 months, at least 12 months, at least 15 months, or atleast 18 months (when stored at a temperature of between about 2 andabout 40° C.).

For example, certain ready-to-administer epinephrine composition had,after storage of at least one month at not less than 25° C., totalimpurities of equal or less than about 0.5% and equal or less than about1% S-isomer content, or had, after storage of at least one month at 25°C. +/−2° C., total impurities of equal or less than about 0.2% and equalor less than about 1.5% S-isomer content, or had, after storage of atleast one month at 25° C. +/−2° C., total impurities of equal or lessthan about 0.5% and equal or less than about 1% S-isomer content, orhad, after storage of at least one month at 25° C. +/−2° C., totalimpurities of equal or less than about 0.3% and equal or less than about0.7% S-isomer content, or had, after autoclaving, total impurities ofequal or less than about 0.5% and equal or less than about 2.0% S-isomercontent, or had, after autoclaving, total impurities of equal or lessthan about 0.2% and equal or less than about 1.5% S-isomer content.

With respect to the sterilization of contemplated formulations it shouldbe appreciated that contemplated formulations may be sterilized usingall known manners of sterilization, including filtration through 0.22micron filters, heat sterilization, autoclaving, radiation (e.g., gamma,electron beam, microwave). Unexpectedly, and as shown in more detailbelow, the inventors have also discovered that contemplated formulationswere heat stable and did not undergo significant isomerization, evenunder conditions of sterilization (exposure to high-pressure saturatedsteam) at 121° C. for at least 5, or at least 10, or at least 15minutes. Thus, terminal sterilization to sterility is possible usingcontemplated compositions and methods.

Based on the unexpected heat stability, the formulations contemplatedherein can also be filtered through a 0.22 micron filter, and filled into a polyethylene, polypropylene or low-density polyethylene containersin a blow-fill-seal (BFS) process. BFS is a form of advanced asepticmanufacturing wherein the container is formed, filled, and sealed in onecontinuous, automated system not requiring human intervention. Theprocess begins with the extrusion of plastic granules in the form of ahot hollow pipe of molten plastic called a parison. The next step is theblow molding of the container with an open top through which thecontainer is filled, all while the plastic remains hot and in a moltenstate. Once filled, the container is hermetically sealed and cooled. Theblow-fill seal process can take several seconds, and contemplatedready-to-administer compositions advantageously are formulated towithstand the temperature and pressure requirements without substantialdegradation of epinephrine (e.g., less than about 5 wt %, less thanabout 3 wt %, less than about 2 wt %, less than about 1 wt %degradation).

Once the epinephrine formulations are filled in large volume polymeric,semi-permeable infusion containers (e.g., BFS container or flexible IVbags), the containers can optionally be layered or covered with asecondary packaging system including an aluminum pouch or other oxygenscavenger. For example, the BFS containers can further be sealed in anoxygen and moisture barrier blister packaging. The blister packaging cancomprise one or more layers, and the one or more layers can includealuminum foil or other oxygen absorber having an oxygen transmissionrate (OTR) as discussed above. Additionally or alternatively, one ormore oxygen absorbers (metal or metal free, organic material) can beincorporated into any portion of the BFS container, the secondarypackaging system, or between the two (e.g., between the BFS containerand the multi-layer packaging) such that the oxygen absorber removes atleast a portion of oxygen from the air surrounding said oxygen-sensitivedrug (e.g., dissolved oxygen in composition and/or oxygen in anyheadspace). A beneficial feature of the oxygen absorber is theabsorbance and removal of oxygen present in the primary packaging and inthe liquid drug itself. Notably, it was found that the oxygen absorberalso removed residual headspace oxygen in the primary packaging and alsodissolved oxygen in the liquid over time, thereby further improvingstability of epinephrine.

For example, the polymeric container may be configured as a flexible bagwith a volume of at least 100 ml, or at least 200 ml, or at least 300ml, or at least 400 ml, or at least 500 ml, or at least 1,000 ml,wherein the polymeric bag may be manufactured from polyvinyl chloride,polyethylene, polypropylene, polyethylene terephthalate, variousco-polymers such as ethylene-propylene copolymers, ethylene vinylacetate, copolyester ether polymers, etc. Such polymeric containers maypreferably, but not necessarily have a reduced oxygen permeability(e.g., where no overwrap is used). These containers or bags may thenpreferably packaged into an overwrap (or other secondary package) thathas one or more additional properties that help maintain stability. Forexample, additional properties include light-absorbing ornon-transparent films to block or at least significantly reduce ingressof light of a wavelength and/or energy sufficient to initiate photolyticdegradation. Other additional properties include reduced oxygenpermeability that can be achieved in a variety of manners, includingmulti-layered polymer and/or metal films that may also include oxygenscavenging materials. For example, a suitable overwrap may comprise apolypropylene base layer that is coupled to a thin aluminum layer (e.g.,thickness between 10 and 50 micrometer), which may be covered by anoriented polyester layer (e.g., commercially available as MEDIFLEX AUAT™from Amcor Flexibles, Gent, Belgium).

The commercially available and concentrated formulation, EpinephrineInjection USP (1 mg/mL) will after dilution in dextrose or dextrose andsodium chloride injections quickly develop colored impurities. Incontrast, and as is shown in more detail below, the antioxidant-free andstorage stable ready-to-administer epinephrine compositions presentedherein are stable over extended periods at room temperature as well asunder refrigeration (e.g., temperature between about 2 and about 8° C.).Impurities resulting from chemical reactions in solution remain withinacceptable limits (e.g., less than about 10 wt %, less than about 5 wt%, less than about 4 wt %, less than about 3 wt %, less than about 2 wt%, less than about 1 wt %) over long term storage (e.g., at least 1month, at least 2 months, at least 3 months, at least 6 months, at least9 months, at least 12 months, at least 18 months.). Therefore,epinephrine formulations of the inventive subject matter can be providedin a ready-to-administer form to avoid the inconvenience associated withdiluting a concentrated small volume epinephrine parenteral formulationinto infusion diluents prior to infusion. The ready-to-administerformulations also eliminate microbial contamination risks andcalculation errors associated with dilution. In certain aspects of theinventive subject matter, contemplated formulations will be available ina range of concentrations commonly required by medical practitioners foremergency restoration of blood pressure in cases of acute hypotension.Thus, ready-to-administer formulations are formulations that can beadministered to a patient in need thereof without prior dilution(typically at the point of care such as a hospital or physician office)from a previously stored solution having a higher epinephrineconcentration. Viewed from a different perspective, theready-to-administer formulations presented herein can be received andstored at a care facility, and then directly used for administrationwithout prior dilution.

The following examples are provided for illustrative purposes only andshould not be interpreted as limiting the present invention.

EXAMPLES

The following examples illustrate some of the experiments leading to theformulations according to the inventive subject matter, however, shouldnot be construed to limit the scope of the claims in any way.

The stability of epinephrine formulation at different pH conditions,ranging from 3.5 to 5.0 was studied at different temperature conditionsof, 25° C., 40° C., and 60° C. All formulations were prepared undersimilar conditions, and formulation details are given in Table 1. Theformulations were analyzed for appearance, pH, and assay. The results ofthe stability studies are presented in Tables 2-5.

For preparation of the solutions, about 90% of the final quantity ofwater was collected in a glass media bottle. Nitrogen (N₂) gas waspurged for about thirty minutes to reduce dissolved oxygen. Sodiumchloride was added and the solution was stirred until a homogenoussolution was obtained. The pH of the bulk solutions was adjusted to pH3.5, 4.0, 4.5, and 5.0 respectively for each formulation compositionusing sufficient quantity of sodium hydroxide or hydrochloric acid.Epinephrine was added and the solution was stirred for approximately 10minutes until a clear solution was formed. Solutions were made up tovolume with water.

TABLE 1 Example 1 Example 2 Example 3 Example 4 RD-043-018A RD-043-018BRD-043-018C RD-043-018D Ingredients Qty/mL (mg) Qty/mL (mg) Qty/mL (mg)Qty/mL (mg) Epinephrine 0.016 0.016 0.016 0.016 Sodium Chloride 9.0 9.09.0 9.0 Disodium Edetate 0.01 0.01 0.01 0.01 Sodium Hydroxide q.s. q.s.q.s. q.s. Hydrochloric Acid q.s. q.s. q.s. q.s. Vehicle q.s. 1 mL q.s. 1mL q.s. 1 mL q.s. 1 mL pH 3.5 4.0 4.5 5.0

TABLE 2 Results for RD-043-018A, pH 3.5 (Example 1) 25° C. ± 2° C./60%RH ± 5% RH 40° C. ± 2° C./75% RH ± 5% RH TEST Initial 3 Day 1 Wk. 2 Wks.3 Wks. 4 Wks. 3 Day 1 Wk. 2 Wks. 3 Wks. 4 Wks Appearance Clear ClearClear Clear Clear Clear Clear Clear Clear Clear Clear colorlesscolorless colorless colorless colorless colorless colorless colorlesscolorless colorless colorless solution solution solution solutionsolution solution solution solution solution solution solution pH 3.583.64 3.62 3.64 3.76 3.69 3.65 3.63 3.63 3.71 3.73 Assay 100.10 99.6798.26 99.60 97.88 99.63 99.43 98.41 98.95 97.35 97.98

For Example 1, it was observed that the formulation was stablephysically, as there was no change in the appearance, pH and assay ofthe formulation at 25° C. and 40° C. at the end of 4 weeks when comparedto initial.

TABLE 3 Results for RD-043-018B, pH 4.0 (Example 2) 25° C. ± 2° C./60%RH ± 5% RH 40° C. ± 2° C./75% RH ± 5% RH TEST Initial 3 Day 1 Wk. 2 Wks3 Wks 4 Wks. 3 Day 1 Wk. 2 Wks. 3 Wks. 4 wks Appearance Clear ClearClear Clear Clear Clear Clear Clear Clear Clear Clear colorlesscolorless colorless colorless colorless colorless colorless colorlesscolorless colorless colorless solution solution solution solutionsolution solution solution solution solution solution solution pH 4.354.30 3.68 4.23 4.34 4.41 4.38 4.41 4.36 4.53 4.34 Assay 99.33 98.0997.69 99.05 96.64 97.65 97.99 98.33 95.90 92.34 79.38

For Example 2, it was observed that the formulation was stablephysically, at 25° C. and 40° C. for 4 weeks as there was no significantchange in the pH, appearance and assay of the formulation.

TABLE 4 Results for RD-043-018C, pH 4.5 (Example 3) 25° C. ± 2° C./60%RH ± 5% RH 40° C. ± 2° C./75% RH ± 5% RH TEST Initial 3 Day 1 Wk. 2 Wks.3 Wks. 4 Wks. 3 Day 1 Wk. 2 Wks. 3 Wks. 4 wks. Appearance Clear ClearClear Clear Slight Slight Clear Clear Slight Slight Slight colorlesscolorless colorless colorless pink brown colorless colorless brown brownbrown solution solution solution solution solution solution pH 4.35 5.174.97 4.89 4.83 4.82 5.03 5.07 5.00 4.50 4.70 Assay 97.22 97.86 99.1289.64 87.53 83.19 97.97 94.87 65.27 66.76 58.52

For example 3, it was observed that the formulation was not stable at pH4.5. A change in the visual appearance was observed at 25° C. and 40° C.conditions. Also the drug formulation showed decreasing assay numberswith increase in the duration of storage as compared to examples 1 and2.

TABLE 5 Results RD-043-018D, pH 5.0 (Example 4) 25° C. ± 2° C./60% RH ±5% RH 40° C. ± 2° C./75% RH ± 5% RH TEST Initial 3 Day 1 Wk. 2 Wks. 3Wks. 4 Wks. 3 Day 1 Wk. 2 Wks. 3 Wks. 4 wks. Appearance Clear ClearClear Clear Slight Slight Light Light Slight Slight Slight colorlesscolorless colorless colorless pink pink brown pink brown brown brownsolution solution solution solution pH 5.15 5.40 5.95 5.01 5.17 5.225.66 5.08 5.22 4.44 4.40 Assay 96.50 99.84 104.33 68.70 73.94 67.45102.01 93.20 59.39 36.63 36.02

For example 4, significant change in the visual appearance and assay wasobserved at 25° C. and 40° C. when compared to Example 1, Example 2 andExample 3. The inventor found that the formulation was more stable at pH3.5-4.0, as compared to pH 4.5 and 5.0. As the pH of the formulation wasincreased, the stability of the formulation decreased.

The presence of atmospheric oxygen promotes the oxidation rate ofepinephrine. Since oxidation frequently involves free radicals, chainreactions occur. Light provides the necessary energy to initiate theoxidation process of epinephrine. Therefore, further experiment wasperformed in Water for Injection as vehicle, EDTA as chelating agent,and controlled overhead space using flexible IV bags which were furtherpackaged in an aluminum foil pouch with an oxygen scavenger (secondarypackaging) or other aluminum pouches containing an oxygen absorbinglayer (secondary packaging) as final packaging. The ready-to-administersolutions of example 5 (Table 6) was prepared according to the aboveprocedure of Example 1-4 with head space oxygen levels controlled (i.e.,replaced by inert gas) during compounding.

All final formulation filling was performed under inert gas atmosphere.To prevent further oxidation degradation, the inventors controlled headspace oxygen in the range of 0.1% to 20%. This was achieved by purginghead space with inert gas as explained above. The formulation solutionwas filled in the flexible IV bags and then placed in an aluminum bagwith an oxygen scavenger (secondary packaging) and sealed. The sealedbags were then kept for stability study at 25° C. and 40° C. The resultsof the stability study of Example 5 are provided in Table 7.

TABLE 6 Example 5 Ingredients Qty/mL (mg) Epinephrine 0.016 SodiumChloride 9.0 EDTA 0.01 Sodium Hydroxide q.s. Hydrochloric Acid q.s.Vehicle q.s. 1 mL pH 4.0 ± 0.2

TABLE 7 Results for RD-043-001 (Example 5) 25° C. ± 2° C./ 40° C. ± 2°C./ 60% RH ± 5% RH 75% RH ± 5% RH TEST Initial 1 Month 3 Months 1 Month3 Months Appearance Clear Clear Clear Clear Clear colorless colorlesscolorless colorless colorless solution solution solution solutionsolution Color L NT 99 99.8 99.9 99.8 Change a NT 0.0 0.0 0.0 0.0 b NT0.0 0.0 0.0 0.0 pH 3.64 3.70 3.68 3.73 4.26 Assay (%) 103.7 102.74101.55 103.11 101.75 Total 0.140 0.325 0.511 0.690 0.703 Impurities (%)NT-Not tested

It was found in Example 5 formulation, for 3 months at 25° C. and 40° C.temperature (accelerated conditions) that epinephrineready-to-administer formulations remained clear and colorless, withoutany signs of precipitation or color change upon storage. The pH of theformulation did not change significantly at different temperatureconditions. There was no significant change in the assay of theformulation, which clearly indicates the formulation is very stable.

Further the inventors studied the effect of autoclaving on the formationof S (+) isomer which is the inactive form in the formulation. Example 6was subjected to autoclaving at 121° C. for 15 minutes. The autoclavedvs non-autoclaved samples were analyzed for assay and related substancesand for the formation of S (+) isomer. Example 6 formulation wasprepared similar to Example 1 and 2. Table 8 shows the composition ofExample 6. The results are shown in Table 9.

TABLE 8 Example 6 Lot # 10358 Lot # 10359 Ingredients Qty/mL (mg) Qty/mL(mg) Epinephrine 0.008 0.032 Sodium Chloride 9.0 9.0 Disodium Edetate0.01 0.01 Sodium Hydroxide q.s. q.s. Hydrochloric Acid q.s. q.s. Waterfor Injection q.s. 1 mL q.s. 1 mL

TABLE 9 Lot # 10358 Lot # 10359 Non- Non- Test Parameters autoclavedAutoclaved autoclaved Autoclaved Assay 100.267 100.779 100.312 103.395 %S form 0.00 2.403 0.00 1.996 Total Impurities 0.23 0.62 0.23 0.37

The inventors therefore concluded that (i) water for injection is a goodvehicle which improves ready-to-administer Epinephrine solutionstability; (ii) at higher pH, the stability of ready-to-administerformulation decreases (iii) controlled head space oxygen levels of <1%helped in improving the stability of the ready-to-administer solution ofEpinephrine. Moreover, stability was remarkably high even at very lowchelator concentration.

In still further experiments, the inventors prepared various epinephrineformulations as shown in Table 10, which included tartrate as a furthercomponent, and exemplary results for the stability of these formulationsare provided in Table 11 (16 mcg/mL, non-terminally sterilized) andTable 12 (16 mcg/mL, terminally sterilized).

TABLE 10 Ingredient Purpose NVK-019 Formulations Epinephrine Active 8mcg/mL 16 mcg/mL 20 mcg/mL 32 mcg/mL 40 mcg/mL pharmaceutical IngredientSodium Isotonicity 9 mg/mL 9 mg/mL 9 mg/mL 9 mg/mL 9 mg/mL chlorideadjusting agent Disodium Metal 0.01 mg/mL 0.01 mg/mL 0.01 mg/mL 0.01mg/mL 0.01 mg/mL Edetate chelator/ dihydrate stabilizer (EDTA) TartaricBuffering 6.6 mcg/mL 13.1 mcg/mL 16.4 mcg/mL 26.2 mcg/mL 32.8 mcg/mLAcid agent Hydrochloric pH Q.S. to adjust pH to 4.0 acid/sodiumadjusting hydroxide agent Water Vehicle Q.S. to 1 mL

TABLE 11 TIME POINT 25° C. ± 2° C./60% RH ± 5% RH 40° C. ± 2° C./75% RH± 5% RH Test Intial 1M 2M 3M 4M 1M 2M 3M 4M Appearance Clear Clear ClearClear Clear Clear Clear Clear Clear Colorless Colorless ColorlessColorless Colorless Colorless Colorless Colorless Colorless solutionsolution solution solution solution solution solution solution solutionColor L 100.9 99.9 100 99.8 99.9 100 100 99.9 96.3 of a 0 0 −0.1 0 0 0 00 0 solution b 0.4 0.1 0 0.1 0.1 0 0 0.1 −1.6 pH 4.24 4.24 4.24 4.144.14 4.25 4.26 4.21 4.23 Dissolved Oxygen (ppm) 0.61 0.66 0.83 0.72 0.790.84 1.41 0.84 0.97 Assay of Epinephrine (%) 98.0 98.1 98.1 98.8 97.099.1 98.7 98.9 96.7 Related RRT-1.24 ND 0.10 0.07 0.10 0.09 0.10 0.090.12 0.13 Substance RRT-1.28 0.07 0.04 ND 0.04 0.03 0.05 0.05 0.08 0.09(%) RRT-1.30 0.13 0.22 0.25 0.06 ND 0.12 0.03 0.09 ND RRT-3.46 0.1 ND NDND ND ND ND ND ND RRT-4.04 ND ND ND ND ND 0.07 ND ND ND Total Impurities(%) 0.3 0.4 0.3 0.2 0.1 0.3 0.2 0.3 0.2 S-Isomer Content (%) 0.2 0.4 0.40.6 0.9 0.9 1.4 2.3 3.1 Note: Impurities ≤0.05% are not reported

TABLE 12 TIME POINT 25° C. ± 40° C. ± Initial 2° C./ 2° C./ Prior toAfter 60% RH ± 75% RH ± Auto- Auto- 5% RH 5% RH 60° C. TEST clavingclaving 1 Month 1 Month 1 week 3 weeks 1 month Appearance Clear ClearClear Clear Clear Clear Clear colorless colorless colorless colorlesscolorless colorless colorless solution solution solution solutionsolution solution solution pH 3.78 3.76 3.91 3.89 3.77 3.75 3.89Dissolved Oxygen (ppm) 1.09 1.74 1.25 1.37 0.77 0.48 0.7 Color L 99.999.9 103.4 103 99.1 100 101.8 of a 0 0 0 0 0.1 0 0.3 solution b −0.1 0−1.4 −1.4 0 0.1 −1.8 Assay of Epinephrine (%) 100.9 100.9 102.1 101.1100.9 101.9 100.8 Related RRT-1.20 0.04 0.06 0.09 0.09 0.15 0.16 0.17Substances RRT-1.24 0.08 0.02 ND ND ND ND ND (%) RRT-1.26 ND 0.07 ND ND0.15 ND ND RRT-1.29 ND ND 0.04 0.05 ND 0.14 0.16 RRT-7.95 ND ND ND ND ND0.06 0.08 Total Imputities (%) 0.1 0.2 0.1 0.1 0.3 0.4 0.4 S-isomercontent (%) 0.3 1.5 0.7 1.4 3.8 8.1 10.2 Note: Impurities ≤0.0.5% arenot reported

Similarly, in additional experiments, the inventors prepared unbufferedepinephrine formulations as shown in Table 13 (without tartrate), andexemplary results for the stability of these formulations are providedin Table 14 (16 mcg/mL, non-terminally sterilized) and Table 15 (16mcg/mL, terminally sterilized).

TABLE 13 Ingredient Purpose NVK-019 Formulations Epinephrine Active 8mcg/mL 16 mcg/mL 20 mcg/mL 32 mcg/mL 40 mcg/mL pharmaceutical IngredientSodium Isotonicity 9 mg/mL 9 mg/mL 9 mg/mL 9 mg/mL 9 mg/mL chlorideadjusting agent Disodium Metal 0.01 mg/mL 0.01 mg/mL 0.01 mg/mL 0.01mg/mL 0.01 mg/mL Edetate chelator/ dihydrate stabilizer (EDTA)Hydrochloric pH Q.S. to adjust pH to 4.0 acid/ adjusting sodium agenthydroxide Water Vehicle Q.S. to 1 mL

TABLE 14 TIME POINT 25° C. ± 2° C./60% RH ± 5% RH 40° C. ± 2° C./75% RH± 5% RH TEST Initial 1M 2M 3M 4M 1M 2M 3M 4M Appearance Clear ClearClear Clear Clear Clear Clear Clear Clear Colorless Colorless ColorlessColorless Colorless Colorless Colorless Colorless Colorless solutionsolution solution solution solution solution solution solution solutionColor of L 100 99.9 100 99.8 99.9 100 100.1 99.9 99.9 solution a 0 0 0 00 0 0 0 −0.1 b −0.3 0 0 0 0.1 0 0 0 0 pH 4.03 4.03 3.90 3.94 3.94 4.044.06 3.98 4.01 Dissolved Oxygen (ppm) 1.52 0.82 1.17 0.91 1.00 0.70 1.220.87 0.74 Assay of Epinephrine (%) 98.7 99.8 98.9 99.4 99.1 99.6 99.199.9 98.1 Related RRT-1.242 ND 0.07 0.12 0.09 0.12 0.07 0.20 0.21 0.25Substances RRT-1.288 ND ND 0.04 ND 0.04 ND 0.11 0.12 0.13 (%) RRT-1.40ND ND ND ND 0.08 ND ND ND ND RRT 1.305 0.15 0.31 0.26 0.11 0.03 0.120.03 0.25 ND RRT-3.463 0.15 ND ND ND ND ND ND ND ND RRT-4.044 ND 0.08 NDND ND 0.09 ND ND ND Total Impurities (%) 0.3 0.5 0.4 0.2 0.3 0.3 0.3 0.60.4 S-Isomer content (%) 0.2 0.3 0.5 0.6 0.8 1.0 1.6 2.5 3.3

TABLE 15 TIME POINT 25° C. ± 40° C. ± 2° C./ 2° C./ Initial 60% RH ± 75%RH ± Prior to After 5% RH 5% RH 60° C. TEST Autoclaving Autoclaving 1month 1 month 1 week 3 weeks 1 month Appearance Clear Clear Clear ClearClear Clear Clear Colorless Colorless Colorless Colorless ColorlessColorless Colorless solution solution solution solution solutionsolution solution pH 4.03 3.93 3.99 4.02 3.95 3.95 3.98 Dissolved Oxygen(ppm) 1.38 1.41 0.95 0.69 0.92 0.68 0.88 Color of L 100 100.3 103.5103.5 100 99.1 103.4 solution a 0 0.1 0 0 0 0.1 0 b 0 −0.1 −1.4 −1.4 00.1 −1.3 Assay of Epinephrine (%) 102.8 101.8 99.9 99.9 100.4 100.6 98.6Related RRT-1.20 0.02 0.04 0.07 0.08 0.09 0.14 0.12 Substances RRT-1.240.10 0.08 0.10 ND ND ND ND (%) RRT-1.29 ND ND 0.05 0.06 ND 0.14 0.12RRT-1.46 0.12 0.11 0.11 0.12 0.11 0.13 0.14 Total impurities (%) 0.2 0.20.3 0.3 0.20 0.4 0.4 S-Isomer content (%) 0.3 0.6 0.7 1.4 2.7 7.8 9.2

Similarly, Tables 16, 17, 18 depict exemplary results for epinephrinecompositions as shown in Table 13, with epinephrine concentrations of 20mcg/mL, 32 mcg/mL, and 40 mcg/mL, respectively, all after terminalsterilization.

TABLE 16 TIME POINT 25° C. ± 40° C. ± 2° C./ 2° C./ 60% RH ± 75% RH ± 5%RH 5% RH 60° C. TEST Initial 2 Wks. 2 Wks. 1 Wk. 2 Wks. Appearance ClearClear Clear Clear Clear Colorless Colorless Colorless ColorlessColorless solution solution solution solution solution pH 4.18 4.06 4.084.03 4.06 DO (ppm) 2.16 0.65 0.84 0.66 0.73 Color L* 100 100.1 100.1100.1 99.9 of a* 0 0 0 0.1 0 solution b* 0.1 −0.1 −0.1 0 0 Assay 100.47699.223 100.116 97.854 99.129 Related RRT-1.219 NR ND ND ND ND SubstancesRRT-1.239 NR 0.055 0.087 0.092 0.110 (%) RRT-1.306 0.063 0.065 0.0880.116 0.137 RRT-1.367 NR NR NR NR NR RRT-1.42 NR ND ND ND ND RRT-1.863ND ND ND 0.50 ND RRT-2.53 ND 0.055 0.058 NR 0.051 RRT-2.93 ND ND ND ND0.060 RRT-3.430 0.097 0.074 0.073 0.109 ND Total Impurities (%) 0.1600.252 0.306 0.367 0.358 S-Isomer Content (%) 1.058 1.205 1.443 4.2036.012

TABLE 17 TIME POINT 25° C. ± 40° C. ± 2° C./ 2° C./ 60% RH ± 75% RH ± 5%RH 5% RH 60° C. TEST Initial 2 Wks. 2 Wks. 1 Wk. 2 Wks. Appearance CCSCCS CCS CCS CCS pH 4.13 4.06 4.05 4.02 4.07 DO (ppm) 2.34 0.90 0.71 0.680.95 Color L* 100.5 100.3 100.3 100 100.1 a* 0 0 0 0 0 b* −0.1 −0.1 0 00 Assay 100.67 100.670 99.575 99.138 100.232 Related RRT-1.113 ND ND ND0.058 ND Substances RRT-1.239 NR NR 0.055 NR 0.085 RRT-1.306 0.077 0.0690.080 0.104 0.121 RRT-1.38 ND ND ND ND 0.093 RRT-1.42/1.40 ND ND ND0.088 ND RRT-1.95 0.172 ND ND ND 0.184 RRT-2.53 NR 0.052 0.058 0.0530.061 RRT-2.93 0.076 ND ND NR 0.063 RRT-3.450 0.135 0.074 ND 0.080 NDTotal Impurities (%) 0.460 0.195 0.193 0.383 0.607 S-Isomer content (%)2.201 2.201 2.419 5.077 7.077

TABLE 18 TIME POINT 25° C. ± 40° C. ± 2° C./ 2° C./ 60% RH ± 75% RH ± 5%RH 5% RH 60° C. TEST Initial 2 Wks. 2 Wks. 1 Wk. 2 Wks. Appearance CCSCCS CCS CCS CCS pH 4.14 4.07 4.07 4.05 4.08 DO (ppm) 1.83 0.97 0.60 0.600.81 Color L* 100.2 100.1 100.3 100 100 a* 0 0 0 0 0.1 b* 0 0 0 0 0.1Assay 98.028 99.880 99.832 98.620 99.326 Related RRT-1.219 0.050 ND NDND ND substances RRT-1.239 ND NR 0.071 0.080 0.084 RRT-1.306 ND 0.0970.116 0.133 0.133 RRT-1.32 0.090 ND ND ND ND RRT-1.42/1.38 ND ND ND ND0.094 RRT-1.863 ND ND ND 0.054 ND RRT-1.91 ND ND ND 0.072 ND RRT-2.580.053 0.07 0.065 0.061 0.062 RRT-2.93 ND ND ND ND ND RRT-3.450 0.1280.047 ND ND ND Total Impurities 0.321 0.214 0.252 0.400 0.432 S-IsomerContent (%) 2.713 3.124 3.362 5.687 7.357

Notably, the above series of experiments demonstrated an unexpectedstability of the low-concentration epinephrine formulations with respectto lack of isomerization (even when terminally sterilized) and withrespect to degradation/oxidation products for both the epinephrine baseand the epinephrine tartrate salt forms. While not wishing to be boundby any particular theory or hypothesis, the inventors contemplate thatthe stability of the epinephrine in the low-concentration formulations(i.e., equal or less than about 50 mcg/mL, or equal or less than about40 mcg/mL, or equal or less than about 32 mcg/mL, or equal or less thanabout 20 mcg/mL, or equal or less than about 16 mcg/mL, or equal or lessthan 8 mcg/mL) was at least in part attributable to thelow-concentration of a chelator (e.g., equal or less than about 30mcg/mL, or equal or less than about 20 mcg/mL, or equal or less thanabout 10 mcg/mL), the specific pH range (e.g., between about 3.65 andabout 4.25, or between about 3.75 and about 4.50) at relatively lowquantities of dissolved O₂ (e.g., equal or less than about 1.8 ppm, orequal or less than about 1.6 ppm, or equal or less than about 1.4 ppm,or equal or less than about 1.2 ppm, or equal or less than about 1.0ppm, or equal or less than about 0.8 ppm). Unless noted otherwise, allresults for terminal sterilization were from materials packaged in apolymer bag and an overpackage, with an oxygen scavenger disposedbetween the polymer bag and the overpackage.

Identification, Assay and Determination of Related Compounds ofEpinephrine in Epinephrine Injection by HPLC-UV: In general, separationof Epinephrine (EPI) and related compounds is performed using a gradientHPLC method with the UV detection. Pentafluorophenylpropyl terminatedsilica is used as a stationary phase for chromatographic analysis. Themobile phase is prepared by mixing an aqueous formate buffer and mixtureof methanol-acetonitrile. EPI is identified based on the retention timeof the major peak in a Sample solution chromatogram as compared to thatfrom a Reference Standard (RS) solution chromatogram. Quantitation ofEPI is accomplished by comparing corresponding peak areas from theSample solution chromatogram and from the RS solution chromatogram.Related compounds are defined by their retention times (RT) based on theRT data for a Peak Identification Mixture. Quantitation of the relatedcompounds is accomplished by comparing the corresponding peak area fromthe Sample Solution chromatogram to the EPI peak area from the RSsolution chromatogram.

The HPLC system was equipped with a quaternary pump or a binary pump, adegasser, auto sampler, a thermo-stated column compartment, and a DADdetector with a 60 mm cell: Agilent 1260 HPLC with a detector having aMax-Light 60 mm Optical Path Length Cell, or equivalent. Mobile Phase Awas 25 mM Ammonium formate buffer pH 3 in water, and Mobile Phase B was90:10 v/v Methanol/Acetonitrile. Chromatographic conditions are shown inTable 16 below.

TABLE 16 HPLC Agilent 1260 HPLC or equivalent Column Supelco DiscoveryHS F5-5 Column, 5 μm, 4.6 × 250 mm Column 35 ± 2° C. Temperature Sample5 ± 5° C. Temperature Injection volume 80.0 μL Flow Rate 1.0 mL/minDetection DAD detector with 60 mm Optical Path Length Cell Spectrum:200-600 nm, resolution 1.0 nm Single channel: 280 nm, resolution 4.0 nmSampling Rate: 5 Hz Mobile Phase 25 mM Ammonium Formate Buffer pH 3 inWater A Mobile Phase 90:10 v/v Methanol/Acetonitrile B Time SolutionSolution Gradient (min) A (%) B (%) 0.0 99.0 1.0 4.0 99.0 1.0 10.0 90.010.0 40.0 50.0 50.0 43.0 50.0 50.0 43.1 99.0 1.0 50.0 99.0 1.0

Epinephrine Assay: The EPI concentration in the Sample Solution, C_(Smp)(mcg/mL), is calculated using Eqs. 1 and 2.

$\begin{matrix}{{C_{WS} = {\frac{W_{WS}}{V_{Stock}} \times \frac{V_{1}}{V_{WS}} \times \frac{M_{1}}{M_{2}} \times P \times 1000\mspace{14mu} {{mcg}/{mg}}}},} & (1) \\{{C_{Smp} = {\frac{R_{Smp}}{R_{WS}} \times C_{WS} \times D}},} & (2)\end{matrix}$

whereC_(WS) is the EPI concentration (mcg/mL) in the working standardsolution on as is basis,W_(WS) is the EPIB RS weight (mg) used to prepare the stock solution forworking standard solution,V_(Stock) is the volume (mL) of the stock solution for working standardsolution,V₁ is the volume (mL) of the stock solution used for dilution in theworking standard solution preparation,V_(WS) is the volume (mL) of the working standard solution solution,M₁ is the molar mass of Epinephrine (183.20 g/mol),M₂ is the molar mass of Epinephrine Bitartrate (333.29 g/mol),P is the decimal purity factor on as is basis for the epinephrinebitartrate reference solution,R_(Smp) is the EPI peak area from the Sample Solution chromatogram andR_(WS) is the average peak area based on the five replicate injectionsof working standard solution.D in Eq. 2 is a dilution factor, which is equal to unity for 8 mcg/mLproducts and to V_(final)/V_(Smp) for products with otherconcentrations, where V_(final) is the final volume of the dilutedsample solution and V_(Smp) is the volume of the sample solution usedfor dilution. D=2 and 4 for 16 mcg/mL and 32 mcg/mL sample solutions,respectively.

The assay result expressed as a percentage of the label claim forEpinephrine base (% LC) is calculated using Eq. 3.

$\begin{matrix}{{{\% \mspace{14mu} {LC}} = {\frac{c_{Smp}}{LC} \times 100\%}},} & (3)\end{matrix}$

whereC_(Smp) is the Sample Solution concentration (mcg/mL) calculatedaccording to Eq. 2, LC is the product label claim expressed using thesame concentration units, i.e. mcg/mL.

Determination of Related Compounds: Concentrations of individualimpurities and degradants, C_(i) (mcg/mL), in a Sample Solution arecalculated using Eq. 4.

$\begin{matrix}{{C_{i} = {\frac{R_{i}}{R_{WS}} \times C_{WS} \times \frac{1}{RRF_{i}} \times D}},} & (4)\end{matrix}$

where,R_(i) is the peak area for i-th Impurity from the Sample Solutionchromatogram,R_(WS) is the average peak area based on the 5 replicate injections ofworking standard solution andRRF_(i) is the relative response factor for i-th Impurity.C_(WS) and D are defined above.

The impurity content (% Imp) can also be expressed as a percent of theLabel Claim (mcg/mL) for Epinephrine base:

$\begin{matrix}{{\% \mspace{14mu} {Imp}} = {\frac{C_{i}}{LC} \times 100\%}} & (5)\end{matrix}$

where,C_(i) is the concentration of individual impurities and degradants(mcg/mL), in the Sample Solution from Eq. 4 and LC is the product labelclaim expressed using the same concentration units, i.e. mcg/mL.

Table 17 depicts exemplary relative retention times and relativeresponse factors for various impurities.

TABLE 17 Compound RRT RRF EPI (Epinephrine) 1 1 DHMA(DL-3,4-Dihydroxymandelic acid) 0.58 1 Imp B (Norepinephrine) 0.69 1 ImpC (Adrenalone) 1.41 3.46 Adrenochrome 1.37 1.38 MNE (Metanephrine) 1.751 NMD (N-Methyldopamine) 1.67 1 MOA (Methoxy Adrenaline) 1.99 1 NE-Imp E(Norepinephrine Impurity E 3.55 1 (4-(Chloroacetyl)-catechol) Imp E(2-(Benzylmethylamino)-3,4- 4.08 1 dihydroxyphenyl)ethanone)

Enantiomeric Purity of Epinephrine in Epinephrine Injection by HPLC-UV:In general, the determination of R- and S-enantiomers of EPI in the drugproduct solution is performed using an isocratic reverse-phase HPLCmethod with UV detection. Separation is achieved using a protein-basedcolumn with functional chiral selectors. The chiral selector isCellobiohydrolase (CBH), which has been immobilized onto sphericalsilica particles. This enzyme preferentially separates compoundscontaining one or more basic nitrogen groups together with one or morehydrogen-accepting or hydrogen-donating groups. The active form of EPIis known to be the R-enantiomer (REPI). The S-enantiomer (SEPI), whichmay be present in the drug product, is quantitated as a percentage ofthe total response, i.e. by comparing the S-peak response to the sum ofthe R- and S-peak responses from a Sample solution chromatogram. TheREPI and SEPI peaks are assigned by comparing their retention times inthe Sample solution chromatogram to those in a standard solutionchromatogram.

The HPLC system was equipped with a pump, a degasser, an autosampler, athermostated column compartment and a PDA detector: Waters Alliancee2695 HPLC or equivalent. The chromatographic column was a DaicelChiralpak CBH™ column, 5 μm, 4.0×100 mm, Sigma-Aldrich, C/N: 58550AST.Mobile Phase A was 10 mM Sodium Phosphate+EDTA buffer at pH 6.0, andMobile Phase B was 100% isopropanol. Chromatographic conditions areshown in Table 18 below.

TABLE 18 HPLC Waters Alliance e2695 HPLC Column Daicel Chiralpak CBH ™column, 5 μm, 4.0 × 100 mm Column Temperature 20° C. ± 2° C. SampleTemperature 5° C. ± 5° C. Injection volume 80 μL Flow Rate 0.7 mL/minDetection Single channel: 280 nm, resolution 6 nm Spectrum: 200-600 nm,resolution 1.2 nm Sampling Rate 5 points/s Filter time Constant: NormalMobile Phase MP A: 10 mM Sodium phosphate + EDTA buffer pH 6.0 MP B:100% IPA Gradient Isocratic run (85% MP A:15% MP B) Run Time 10 min

Determination of percentage of S-epinephrine: The percentage of SEPI, %S, in a Sample solution is calculated using Eq. 6.

$\begin{matrix}{{{\% \mspace{14mu} S} = {\frac{A_{SEPI}}{A_{SEPI} + A_{REPI}} \times 100\%}},} & (6)\end{matrix}$

where A_(SEPI) is the SEPI peak area from the sample chromatogram,A_(REPI) is the REPI peak area from the same sample chromatogram.

Therefore, various exemplary formulations of the inventive subjectmatter have been provided. The formulations of the inventive subjectmatter can be administered according to any suitable dosing schedule,which can vary depending on the condition to be treated.

The optimum therapeutically effective amount of a drug is the amount ofthe drug in the composition that will yield the most effective resultsin terms of efficacy of treatment in a given subject. This amount canvary depending upon a variety of factors, including but not limited tothe physiological condition of the subject (including age, sex, diseasetype and stage, general physical condition, responsiveness to a givendosage, and type of medication), the nature of the pharmaceuticallyacceptable carrier or carriers in the formulation, and the route ofadministration. One skilled in the clinical and pharmacological artswill be able to determine a therapeutically effective amount throughroutine experimentation, for instance, by monitoring a subject'sresponse to administration of a compound and adjusting the dosageaccordingly. For additional guidance, see Remington: The Science andPractice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA,USA) (2000).

The pharmaceutical forms suitable for injectable use include sterilesolutions, dispersions, emulsions, and sterile powders. The final formshould be stable under conditions of manufacture and storage.Furthermore, the final pharmaceutical form should be protected againstcontamination and should, therefore, be able to inhibit the growth ofmicroorganisms such as bacteria or fungi. The ready-to-administerformulations should also be able to pass readily through an injectiondevice such as a hollow needle.

Depending on the particular purpose, it should also be recognized thatcontemplated compositions may be combined (in vivo, or in a therapeuticformulation or administration regimen) with at least one othertherapeutically active agent to additively or synergistically provide atherapeutic or prophylactic effect.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. Moreover, where the term ‘about’ is used inconjunction with a numeral, a range of that numeral +/−10%, inclusive,is contemplated. In some embodiments, the numerical parameters should beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques. Notwithstanding that thenumerical ranges and parameters setting forth the broad scope of someembodiments of the invention are approximations, the numerical valuesset forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The discussion herein provides example epinephrine compositions andmethods of the inventive subject matter. Although each embodimentrepresents a single combination of inventive elements, the inventivesubject matter is considered to include all possible combinations of thedisclosed elements. Thus if one embodiment comprises elements A, B, andC, and a second embodiment comprises elements B and D, then theinventive subject matter is also considered to include other remainingcombinations of A, B, C, or D, even if not explicitly disclosed.

It should be apparent, however, to those skilled in the art that manymore modifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of thedisclosure. One skilled in the art will recognize many methods andmaterials similar or equivalent to those described herein, which couldbe used in the practice of the present invention. Indeed, the presentinvention is in no way limited to the methods and materials described.

Moreover, in interpreting the disclosure all terms should be interpretedin the broadest possible manner consistent with the context. Inparticular the terms “comprises” and “comprising” should be interpretedas referring to the elements, components, or steps in a non-exclusivemanner, indicating that the referenced elements, components, or stepscan be present, or utilized, or combined with other elements,components, or steps that are not expressly referenced.

What is claimed is:
 1. A method of producing a sterile and storagestabile ready-to-inject epinephrine composition, comprising: combiningan aqueous pharmaceutically acceptable carrier with epinephrine and ametal ion chelator such that the epinephrine has a concentration ofequal or less than 0.07 mg/ml, wherein the metal ion chelator is presentin the composition at a concentration of between about 1 and 50 ug/ml,and wherein substantially all of the epinephrine is an R-isomer;adjusting the pH of the ready-to-inject epinephrine composition to a pHof between 3.0-4.7; packaging the ready-to-inject epinephrinecomposition into a container under an inert gas; and autoclaving theready-to-inject epinephrine composition to sterility.
 2. The method ofclaim 1 wherein sterilization increases S-isomer content by no more than1.5%.
 3. The method of claim 1 wherein sterilization increases S-isomercontent by no more than 0.5%.
 4. The method of claim 1 whereinsterilization increases total impurities content by no more than 0.5%.5. The method of claim 1 wherein sterilization increases totalimpurities content by no more than 0.3%.
 6. The method of claim 1wherein the ready-to-inject epinephrine composition has, aftersterilization and storage of at least one month at 25° C., totalimpurities of equal or less than 0.7% and equal or less than 2% S-isomercontent.
 7. The method of claim 1 wherein the ready-to-injectepinephrine composition has, after sterilization and storage of at leastone month at 25° C., total impurities of equal or less than 0.5% andequal or less than 1% S-isomer content.
 8. The method of claim 1 furthercomprising a step of including a buffer at a concentration of between1-25 mM into the composition.
 9. The method of claim 1 wherein thecomposition comprises dissolved oxygen in an amount of equal or lessthan 1.5 ppm O₂.
 10. A sterile storage stable ready-to-injectepinephrine composition, comprising: an aqueous pharmaceuticallyacceptable carrier containing epinephrine and a metal ion chelator;wherein the epinephrine is present at a concentration of equal or lessthan 0.07 mg/ml, wherein substantially all of the epinephrine is anR-isomer, and wherein the composition has a pH of between 3.0-4.7; andwherein the ready-to-inject epinephrine composition has, after storageof at least one month at 25° C., total impurities of equal or less than0.7% and equal or less than 2% S-isomer content.
 11. The composition ofclaim 10, wherein the epinephrine is present at a concentration of equalor less than 0.04 mg/ml.
 12. The composition of claim 11, wherein thecomposition has, after storage of at least one month at 25° C., totalimpurities of equal or less than 0.5%.
 13. The composition of claim 10,wherein the epinephrine is present at a concentration of equal or lessthan 0.02 mg/ml.
 14. The composition of claim 13, wherein thecomposition has, after storage of at least one month at 25° C., totalimpurities of equal or less than 0.5%.
 15. The composition of claim 10,wherein the composition is an autoclaved sterile composition, andwherein the S-isomer content after autoclaving and after storage of atleast one month at 25° C. is equal or less than 1.3%.
 16. Thecomposition of claim 10, wherein the composition is an autoclavedsterile composition, and wherein the S-isomer content after autoclavingand after storage of at least one month at 25° C. is equal or less than0.7%.
 17. The composition of claim 10, wherein the composition comprisesdissolved oxygen in an amount of equal or less than 1.5 ppm O₂.
 18. Thecomposition of claim 10, wherein the composition further comprises abuffer at a concentration of between 1-25 mM.
 19. The composition ofclaim 10, wherein the composition, after storage of at least one monthat 25° C., changes pH no more than 0.1 pH units.
 20. The composition ofclaim 10, wherein the composition comprises the metal ion chelator at aconcentration of between about 1 and 50 μg/ml.